PROJECT SUMMARY There has been a need to improve depression pharmacotherapies for several decades. Despite this, new treatment advances have been slow to progress, or have failed to reach clinical practice. Perhaps the most impactful advance, has been the discovery that subanesthetic ketamine can rapidly alleviate treatment- resistant major depression. While it is known that ketamine exerts its anesthetic effects through N-methyl-D- aspartate receptor (NMDAR) antagonism, it is unclear as to whether this is its antidepressant mechanism of action. Our lab has previously shown that ketamine is rapidly converted into various metabolites, and at least one of these, (2R,6R)-hydroxynorketamine (HNK), retains the rapid antidepressant-like preclinical properties of ketamine, but lacks its adverse effects given its low affinity to inhibit the NMDAR. My data reveal that (2R,6R)- HNK promotes a rapid potentiation of ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- mediated synaptic transmission, through a concentration-dependent, NMDAR-independent, and synapse- selective increase in glutamate release probability at Schaffer collateral (SC)-CA1 synapses (Riggs et al., 2019, Neuropsychopharmacology). My preliminary data suggest that the presynaptic effects of (2R,6R)-HNK require protein kinase activity, and presynaptic calcium channel influx. Consistent with this, studies have shown that (2R,6R)-HNK leads to a rapid accumulation of intracellular cyclic adenosine monophosphate (cAMP), which converges with the actions of locally elevated calcium to regulate protein kinase activity. Additionally, the behavioral effects of (2R,6R)-HNK converge with a mechanism downstream of presynaptic glutamate auto- receptors that inhibit the production of cAMP, whereas cAMP triggers the release of brain-derived neurotrophic factor (BDNF), which is also required for (2R,6R)-HNK to exert its behavioral effects. Thus, I hypothesize that (2R,6R)-HNK exerts its rapid synaptic potentiation through an acute increase in cAMP-dependent, presynaptic BDNF-TrkB signaling. I will test this hypothesis with three specific aims, using both ex vivo and in vivo approaches. First, I will use acute slice electrophysiology to determine whether the presynaptic effects of (2R,6R)-HNK are cAMP-dependent, and engages its downstream target, protein kinase A (PKA). Using biochemistry, I will verify changes in post-recording presynaptic cAMP, as well as phosphorylation changes in proteins downstream of PKA, known to actively participate in glutamate release. Second, I will test the role of BDNF-TrkB signaling in the presynaptic actions of (2R,6R)-HNK, and assess phosphorylation changes in TrkB and its presynaptic downstream targets. Lastly, I will use in vivo fiber photometry to determine if (2R,6R)-HNK improves learning in a SC-CA1-dependent task by increasing the strength of synaptic transmission at SC-CA1 synapses in a BDNF-dependent manner. My experiments will test the role of presynaptic signal transduction in the acute mechanism of action of (2R,6R)-HNK, which will advance our understanding of how presynaptic plasticity gives rise to sustained adaptations in synaptic transmission and behavior.